The present invention pertains to antibacterial and antimicrobial agents, in particular, the present invention provides methods of synthesizing and screening compounds that are bacterial nicotinamide adenine dinucleotide (NAD) synthetase enzyme inhibitors. The present invention also provides novel compounds that inhibit bacterial NAD synthetase enzyme. The invention also provides libraries of compounds that comprise bacterial NAD synthetase enzyme inhibitors. Further, the present invention provides compounds that exhibit therapeutic activity as antibacterial agents, antimicrobial agents and broad spectrum antibiotics. Still further, the invention provides methods of treating a mammal with bacterial NAD synthetase enzyme inhibitor compounds. The present invention also provides novel disinfecting agents.
Drug-resistant infectious bacteria, that is, bacteria that are not killed or inhibited by existing antibacterial and antimicrobial compounds, have become an alarmingly serious worldwide health problem. (E. Ed. Rubenstein, Science, 264, 360 (1994)). In fact, a number of bacterial infections may soon be untreatable unless alternative drug treatments are identified.
Antimicrobial or antibacterial resistance has been recognized since the introduction of penicillin nearly 50 years ago. At that time, penicillin-resistant infections caused by Staphylococcus aureus rapidly appeared. Today, hospitals worldwide are facing unprecedented crises from the rapid emergence and dissemination of microbes resistant to one or more antimicrobial and antibacterial agents commonly in use today. As stated in the Fact Sheet on Antimicrobial Resistance of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, several strains of antibiotic-resistant bacteria are now emerging and are becoming a threat to human and animal populations, including those summarized below:
1) Strains of Staphylococcus aureus resistant to methicillin and other antibiotics are endemic in hospitals. Infection with methicillin-resistant S. aureus (MRSA) strains may also be increasing in non-hospital settings. Vancomycin is the only effective treatment for MRSA infections. A particularly troubling observation is that S. aureus strains with reduced susceptibility to vancomycin have emerged recently in Japan and the United States. The emergence of vancomycin-resistant strains would present a serious problem for physicians and patients.
2) Increasing reliance on vancomycin has led to the emergence of vancomycin-resistant enterococci (VRE), bacteria that infect wounds, the urinary tract and other sites. Until 1989, such resistance had not been reported in U.S. hospitals. By 1993, however, more than 10 percent of hospital-acquired enterococci infections reported to the Centers for Disease Control (xe2x80x9cCDCxe2x80x9d) were resistant.
3) Streptococcus pneumoniae causes thousands of cases of meningitis and pneumonia, as well as 7 million cases of ear infection in the United States each year. Currently, about 30 percent of S. pneumoniae isolates are resistant to penicillin, the primary drug used to treat this infection. Many penicillin-resistant strains are also resistant to other antimicrobial or antibacterial drugs.
4) Strains of multi-drug resistant tuberculosis (MDR-TB) have emerged over the last decade and pose a particular threat to people infected with HIV. Drug-resistant strains are as contagious as those that are susceptible to drugs. MDR-TB is more difficult and vastly more expensive to treat, and patients may remain infectious longer due to inadequate treatment. Multi-drug resistant strains of Mycobacterium tuberculosis have also emerged in several countries, including the U.S.
5) Dianheal diseases cause almost 3 million deaths a year, mostly in developing countries, where resistant strains of highly pathogenic bacteria such as Shigella dysenteriae, Campylobacter, Vibrio cholerae, Escherichia coli and Salmonella are emerging. Furthermore, recent outbreaks of Salmonella food poisoning have occurred in the United States. A potentially dangerous xe2x80x9csuperbugxe2x80x9d known as Salmonella typhimurium, resistant to ampicillin, sulfa, streptomycin, tetracycline and chloramphenicol, has caused illness in Europe, Canada and the United States.
In addition to its adverse effect on public health, antimicrobial or antibacterial resistance contributes to higher health care costs. Treating resistant infections often requires the use of more expensive or more toxic drugs and can result in longer hospital stays for infected patients. The Institute of Medicine, a part of the National Academy of Sciences, has estimated that the annual cost of treating antibiotic resistant infections in the United States may be as high as $30 billion.
Given the above, it would be highly desirable to develop novel antibacterial and antimicrobial agents that act by different mechanisms than those agents in use currently. Further, it would be desirable to be able to synthesize such novel compounds. It would also be desirable to develop libraries of compounds that exhibit inhibitory bacterial NAD synthetase activity. Such new agents would be useful to counteract antibiotic resistant strains of bacteria and other types of harmful microbes. It would be even more desirable to develop antibacterial agents that inhibit or block essential bacterial metabolic mechanisms, to result in bacterial death or deactivation, without also affecting the essential metabolic activities of a mammalian host. That is, it would be desirable to develop antibacterial agents that preferentially attack bacteria and other microbes and kill or deactivate the harmful organism without causing any attendant undesirable side effects in a human or animal patient. It would also be desirable to develop methods of rapidly screening potential new antimicrobial and antibacterial agents. It would also be desirable to develop novel disinfecting agents.
In one aspect, the invention provides a NAD synthetase inhibitor compound of the formula: 
Still further, the invention provides a bacterial NAD synthetase enzyme inhibitor compound of the structure: 
In yet a further embodiment, the invention provides a bacterial NAD synthetase enzyme inhibitor of the formula: 
In a further aspect, the invention provides a bacterial NAD synthetase enzyme inhibitor compound, having Structure 2: 
wherein n is an integer of from 1 to 12, R1-R7 each, independently, is an H, an unsubstituted or a substituted cyclic or aliphatic group, a branched or an unbranched group, wherein the linker is a cyclic or aliphatic, branched or an unbranched alkyl, alkenyl, or an alkynyl group and wherein the linker may also contain heteroatoms.
In yet another aspect, the invention provides a bacterial NAD synthetase enzyme inhibitor compound, having Structure 4: 
wherein X is a C, N, O or S within a monocyclic or bicyclic moiety, A and B represent the respective sites of attachment for the linker, n is an integer of from 1 to 12, R1-R7 each, independently, is an H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group, wherein the linker is a saturated or unsaturated cyclic group or an aliphatic branched or unbranched alkyl, alkenyl or alkynyl group, and wherein the linker may also contain heteroatoms.
Further, the invention provides a method of treating or preventing a microbial infection in a mammal comprising administering to the mammal a treatment effective or treatment preventive amount of a bacterial NAD synthetase enzyme inhibitor compound. Still further, a method is provided of killing a prokaryote with an amount of prokaryotic NAD synthetase enzyme inhibitor to reduce of eliminate the production of NAD whereby the prokaryote is killed. Moreover, a method is provided of decreasing prokaryotic growth, comprising contacting the prokaryote with an amount of a prokaryotic NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD whereby prokaryotic growth is decreased. Further provided is a disinfectant compound wherein the compound comprises a bacterial NAD synthetase enzyme inhibitor. Still further, the invention provides a method of disinfecting a material contaminated by a microbe, comprising contacting a contaminated material with a bacterial NAD synthetase enzyme inhibitor compound in an amount sufficient to kill or deactivate the microbe.
In yet another aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating 5-nitroindole with 6-bromohexyl acetate to form a 6-[N-(5-nitroindolyl)]hexyl acetate; b. hydrolyzing the 6-[N-(5-nitroindolyl)]hexyl acetate to form N-(5-nitroindolyl)hexan-1-ol; c. esterifying the 6-[N-(5-nitroindolyl)]hexan-1-ol with nicotinic acid to form 6-[N-(5-nitroindolyl)]hexyl nicotinate; and d. N-methylating the 6-[N-(5-nitroindolyl)]hexyl nicotinate.
Further, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a alkylating 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol; b. reacting the indole alkyl alcohol with the appropriate reagent to form an indole alkyl ester, and c. N-methylating the indole alkyl ester.
Moreover, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. reacting indole carboxylic acid with the appropriate reagent to provide an indole carboxylate methyl ester or an indole benzyl carboxylate ester; b. N-alkylating the indole carboxylate methyl ester or the indole carboxylate benzyl ester with bromoalkyl acetate; c. reacting the material from step b above with the appropriate reagent to form an indolealkyl alcohol; d. coupling the indolealkyl alcohol with an aromatic amine; and e. reacting the indolealkyl alcohol with the appropriate reagent to convert the methyl or benzyl indolecarboxylate to the respective indole carboxylic acids.
In another aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline; b. reacting the 2-bromo-R21-substituted-aniline or the 2-bromo-R2-substituted-aniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol; d. quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with methansulfonyl chloride to provide an indole mesylate; and f. reacting the indole mesylate with a carboxylic acid to form an indole ester.
Still further, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline; b. reacting the 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline using a Heck coupling reaction to form an alkyne-substituted aniline; c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol; d. quaternizing the indole alcohol with an amine; e. reacting the indole alcohol with triflouromethylsulfonic anhydride to provide a triflate; and f. reacting the indole triflate with an amine to form an indole alkylammonium product.
In a further aspect, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of: a. alkylating a phenol with 7-bromo-1-heptanol to provide 7-(phenyloxy)-1-heptanol; b. mesylating 7-(phenyloxy)-1-heptanol to provide 7-(phenyloxy)-1-heptyl methanesulfonate; c. esterifying 7-(phenyloxy)-1-heptyl-methanesulfonate to provide 7-(phenyloxy)-1-heptyl nicotinate; and d. n-methylating 7-(phenyloxy)-1-heptyl nicotinate to provide [7-(phenyloxy)-1-heptyl-(N-methyl)nictotinate]iodide.
In yet another aspect, the invention provides a method of generating a library comprising at least one bacterial NAD synthetase enzyme inhibitor compound comprising the steps of: a. obtaining the crystal structure of a bacterial NAD synthetase enzyme; b. identifying one or more sites of catalytic activity on the NAD synthetase enzyme; c. identifying the chemical structure of the catalytic sites on the NAD synthetase enzyme; d. selecting one or more active molecules that will demonstrate affinity for at least one of the catalytic sites on the NAD synthetase enzyme; f. synthesizing one or more dimeric compounds comprised of at least one active molecule wherein the active molecule compound are joined by means of n linker compounds and wherein n is an integer of from 1 to 12, and g. screening the one or more compounds for NAD synthetase inhibitor activity.
In a further aspect of the invention herein, a method is provided for the in vitro screening a compound for bacterial NAD synthetase enzyme inhibitory activity comprising the steps of: a. preparing a bacterial NAD synthetase enzyme solution from pure bacterial NAD synthetase enzyme mixed with a suitable buffer; b. contacting the bacterial NAD synthetase enzyme solution with a test compound; and c. measuring the rate of the enzyme-catalyzed reaction between the NAD synthetase enzyme and the test compound, wherein the rate of the enzyme catalyzed reaction comprises a measure of bacterial NAD synthetase enzyme inhibitory activity.
Additional advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included herein.
Before the present methods, compounds, compositions and apparatuses are disclosed and described it is to be understood that this invention is not limited to the specific synthetic methods described herein. It is to be further understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise.
Ranges may be expressed herein as from xe2x80x9caboutxe2x80x9d one particular value, and/or to xe2x80x9caboutxe2x80x9d another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent xe2x80x9cabout,xe2x80x9d it will be understood that the particular value forms another embodiment.
Throughout this application, where a chemical diagram has a straight line emanating from a chemical structure, such a line represents a CH3 group. For example, in the following diagram: 
o-methylbenzoic acid is represented.
The term xe2x80x9calkylxe2x80x9d as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. The term xe2x80x9ccycloalkylxe2x80x9d intends a cyclic alkyl group of from three to eight, preferably five or six carbon atoms.
The term xe2x80x9calkoxyxe2x80x9d as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an xe2x80x9calkoxyxe2x80x9d group may be defined as xe2x80x94OR where R is alkyl as defined above. A xe2x80x9clower alkoxyxe2x80x9d group intends an alkoxy group containing from one to six, more preferably from one to four, carbon atoms.
The term xe2x80x9calkylenexe2x80x9d as used herein refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene (xe2x80x94CH2xe2x80x94), ethylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94), propylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94), 2-methylpropylene [xe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94], hexylene [xe2x80x94(CH2)6xe2x80x94] and the like. The term xe2x80x9ccycloalkylenexe2x80x9d as used herein refers to a cyclic alkylene group, typically a 5- or 6-membered ring.
The term xe2x80x9calkenexe2x80x9d as used herein intends a mono-unsaturated or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present.
The term xe2x80x9calkynylxe2x80x9d as used herein refers to a branched or unbranched unsaturated hydrocarbon group of 1 to 24 carbon atoms wherein the group has at least one triple bond.
The term xe2x80x9ccyclicxe2x80x9d as used herein intends a structure that is characterized by one or more closed rings. As further used herein, the cyclic compounds discussed herein may be saturated or unsaturated and may be heterocyclic. By heterocyclic, it is meant a closed-ring structure, preferably of 5 or 6 members, in which one or more atoms in the ring is an element other than carbon, for example, sulfur, nitrogen, etc.
The term xe2x80x9cbicyclicxe2x80x9d as used herein intends a structure with two closed rings. As further used herein, the two rings in a bicyclic structure can be the sane or different. Either of the rings in a bicyclic structure may be heterocyclic.
By the term xe2x80x9ceffective amountxe2x80x9d of a compound as provided herein is meant a sufficient amount of the compound to provide the desired treatment or preventive effect. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact xe2x80x9ceffective amount.xe2x80x9d However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation. It is preferred that the effective amount be essentially non-toxic to the subject, but it is contemplated that some toxicity will be acceptable in some circumstances where higher dosages are required.
By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compounds of the invention without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
As used herein, xe2x80x9cNAD synthetase enzymexe2x80x9d is defined as the enzyme that catalyzes the final reaction in the biosynthesis of NAD, namely, the transformation of NaAD into NAD. As used herein, the term xe2x80x9ccatalytic sitesxe2x80x9d are defined as those portions of the NAD synthetase enzyme that bind to substrates, and cofactors, including nictonic acid dinucleotide (NaAD), NAD, adenosine triphosphate (ATP), adenosine monophosphate (AMP), pyrophosphate, magnesium and ammonia in bacteria or microbes. The term xe2x80x9creceptor sitexe2x80x9d or xe2x80x9creceptor subsitexe2x80x9d relates to those portions of the bacterial NAD synthetase enzyme in which the bacterial NAD synthetase enzyme inhibitors disclosed herein are believed to bind. For the purposes of this disclosure, the terms xe2x80x9ccatalytic site,xe2x80x9d xe2x80x9creceptor sitexe2x80x9d and xe2x80x9creceptor subsitexe2x80x9d may be used interchangeably.
As used herein, the terms xe2x80x9clibraryxe2x80x9d and xe2x80x9clibrary of compoundsxe2x80x9d denote an intentionally created collection of differing compounds which can be prepared by the synthetic means provided herein or generated otherwise using synthetic methods utilized in the art. The library can be screened for biological activity in any variety of methods, such as those disclosed below herein, as well as other methods useful for assessing the biological activity of chemical compounds. One of skill in the art will recognize that the means utilized to generate the libraries herein comprise generally combinatorial chemical methods such as those described in Gallop, et al, xe2x80x9cApplications of Combinatorial Techniques to Drug Discovery,xe2x80x9d xe2x80x9cPart 1 Background and Peptide Combinatorial Libraries,xe2x80x9d and xe2x80x9cPart 2: Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions,xe2x80x9d J. Med. Chem., Vol. 37(1994) pp. 1233 and 1385. As used herein, the terms xe2x80x9ccombinatorial chemistryxe2x80x9d or xe2x80x9ccombinatorial methodsxe2x80x9d are defined as the systematic and repetitive, covalent connection of a set of different xe2x80x9cbuilding blocksxe2x80x9d of varying structure, such as the active molecules disclosed herein, to provide a large array of diverse molecular entities. As contemplated herein, the large array of diverse molecular entities together form the libraries of compounds of the invention.
As used herein, the term xe2x80x9cantibacterial compoundxe2x80x9d denotes a material that kills or deactivates bacteria or microbes so as to reduce or eliminate the harmful effects of the bacteria on a subject or in a system. Such materials are also known in the art as xe2x80x9cbacteriostatic agentsxe2x80x9d or xe2x80x9cbacteriocidal agents.xe2x80x9d The bacteria so affected can be gram positive, gram negative or a combination thereof The terms xe2x80x9cantimicrobial compoundxe2x80x9d and xe2x80x9cbroad spectrum antibioticxe2x80x9d denote a material that kills or deactivates a wide variety of microbes, including, but not limited to, one of more of, gram positive or gram negative bacteria, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus viridans, Enterococcus, anaerobic Streptococcus, Pneumococcus, Gonococcus, Meningococcus, Mima, Bacillus anthracis, C. diphtheriae, List. monocytogenes, Streptobacillus monohiliformis, Erysipelothrix insidiosa, E. coli, A. aerogenes, A. faecalis, Proteus mirabilis, Pseudomonas aeruginosa, K. pneumoniae, Salmonella, Shigella, H. influenzae, H. ducreyi, Brucella, Past. pestis, Past. tularensis, Past. muiltocida, V. comma, Actinobacillus mallei, Pseud. pseudomallei, Cl. tetani, Bacteroides, Fusobacterium fusiforme, M. tuberculosis, atypical niycobacteria, Actinomyces israelii, Nocardia, T. pallidum, T. pernue, Borrelia recurrentis, Peptospira, Rickettsia, and Mycoplasma pneumoniae. 
In accordance with the desirability for developing improved antibacterial and antimicrobial agents, with the invention herein novel compounds have been identified that inhibit bacterial NAD synthetase enzymatic activity. Such activity translates into effectiveness as bacteriocidal agents, as well as effectiveness as a broad spectrum antibiotic materials. Novel compounds have been developed that inhibit a previously unrecognized target in prokaryotic organisms, such as bacteria, to block essential biological function and thereby cause bacterial death or deactivation of bacteria or other microbes. Specifically, the invention herein has identified an enzyme found in both gram positive and gram negative bacteria, NAD synthetase enzyme, which can be utilized as a target for drug design to provide protection from and/or treatment for bacterial and other microbial infections.
The NAD synthetase enzyme catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Bacterial NAD synthetase is an ammonia-dependent amnidotransferase belonging to a family of xe2x80x9cN-typexe2x80x9d ATP pyrophosphatases; this family also includes asparagine synthetase and argininosuccinate synthetase. NAD synthetase enzyme catalyzes the last step in both the de novo and salvage pathways for NAD+ biosynthesis, which involves the transfer of ammonia to the carboxylate of nicotinic acid adenine dinucleotide (NAAD) in the presence of ATP and Mg+2. The overall, reaction is illustrated in Scheme 1. 
Unlike eukaryotic NAD synthetase e.g., that found in mammals, which can utilize glutamine as a source of nitrogen, prokaryotic NAD synthetase in bacteria utilizes ammonia as the sole nitrogen source. Through x-ray crystallography and other methods, the invention has identified marked differences in the structures of eukaryotic and prokaryotic forms of the NAD synthetase enzyme. For example, B. subtilis NAD synthetase enzyme, which in the invention has been crystallized and used in the drug design methodologies herein, is a dimeric material with molecular weight around 60,500. In marked contrast, the eukaryotic form of NAD synthetase found in mammals is multimeric and has a molecular weight of at least 10 times larger.
By utilizing the significant differences between the eukaryotic and prokaryotic forms of NAD synthetase enzyme, the invention herein provides novel compounds that can be utilized as antibacterial and antimicrobial agents that specifically target the prokaryotic NAD synthetase enzyme without also effecting a mammalian host. With the invention herein, it has been found that by specifically inhibiting bacterial NAD synthetase enzymatic activity, bacteria can be deprived of the energy necessary to thrive and replicate. Accordingly, through the invention disclosed and claimed herein, antibacterial and antimicrobial drugs have been developed that preferentially attack the bacteria to kill or deactivate it so as to reduce or eliminate its harmful properties, without appreciably affecting mammalian NAD synthetase enzymatic activity at the same dosage.
Furthermore, novel methods are provided that allow the rapid screening of compounds for bacterial NAD synthetase enzyme inhibitory activity. Moreover, the invention provides methods of treating microbial infections in a subject. Because of the differences in structure between bacterial and mammalian NAD synthetase enzyme, it would not be expected that the compounds of the invention would inhibit or otherwise affect mammalian NAD synthetase enzyme in the same manner as the compounds act on bacteria.
Without being bound by theory, through chemical analysis and x-ray crystallography methods, at least two separate catalytic subsites on the bacterial NAD synthetase enzyme in which it is possible to bind at least one or more small molecules (xe2x80x9cactive moleculesxe2x80x9d) have been characterized. These sites are illustrated below by the cartoon in FIG. 2.
FIG. 2: CATALYTIC SITES IN BACTERIAL NAD SYNTHETASE ENZYME 
Because of the specific structure of these catalytic sites, it has been determined that it is possible to identify small molecules that will demonstrate affinity for at least one of the sites. Small molecules of the proper configuration, the configuration being determined by the structure of the catalytic site(s), will bind with a receptor site or sites on the bacterial NAD synthetase enzyme, thereby blocking the catalytic activity of the enzyme. FIG. 4 illustrates via cartoon a bacterial NAD synthetase enzyme in which the catalytic sites are blocked by an example of a compound of the present invention.
FIG. 4: BACTERIAL NAD SYNTHETASE ENZYME WITH BLOCKED CATALYTIC/RECEPTOR SITES 
Under such circumstances, it is hypothesized that spore-forming bacteria will be unable to undergo germination and outgrowth, and the essential cellular respiratory functions of the vegetative bacteria will be halted, thereby causing cellular death or deactivation, e.g., gram positive and gram negative bacteria and other microbes will be killed or prevented from undergoing growth. Accordingly, the invention has found that compounds that exhibit inhibitory activity against the bacterial NAD synthetase enzyme will also exhibit therapeutic activity as antibacterial and antimicrobial compounds, as well as broad spectrum antibiotic materials.
With the invention herein it has been surprisingly found that it is possible to synthesize novel tethered dimeric compounds that will exhibit activity as bacterial NAD synthetase enzyme inhibitors. By linking one or more active molecules through a linker molecule, one or more ends of the tethered dimer can bind in the respective receptor sites or subsites to thereby render the bacterial NAD synthetase enzyme inactive. When more than one active molecule is used, each active molecule can be the same or different. The term xe2x80x9cactive moleculesxe2x80x9d as used herein refers to small molecules that may be used alone or tethered together through a linker (tether) fragment to form a tethered dimeric compound.
In the present invention, the active molecules are comprised of substituent groups as hereinafter disclosed that will bind with at least one of the receptor sites in bacterial NAD synthetase enzyme. In the invention herein one or more active molecules are tethered together to form a dimeric molecule that is capable of inhibiting the bacterial NAD synthetase enzyme.
Further, in this invention it has been found that, under some circumstances, different active molecules will be more likely to bind to different locations in the receptor site of a bacterial NAD synthetase enzyme because of the differing chemical make-up of each of these sites. Therefore, in one embodiment, it is beneficial to tether at least two different active molecules to each other wherein each active molecule demonstrates selective affinity for a different subsite in the receptor. Using the tethered dimers herein it is possible to drastically enhance the potency of NAD synthetase enzyme inhibition, as compared to blocking a single site on the bacterial NAD synthetase enzyme. As used herein, the term xe2x80x9cselective affinityxe2x80x9d means that the active molecule shows enhanced tendency to bind with one subsite with the receptor in the bacterial NAD synthetase enzyme because of a chemical complementarity between the receptor subsite and the active molecule. A tethered dimer compound is illustrated in Scheme 2 below. 
In one embodiment, a dimeric inhibitor compound will bind with, for example, the sites of catalytic activity on the bacterial NAD synthetase enzyme, thereby preventing the production of NAD/NADH by the bacteria. As an additional surprising finding in this invention, it has been determined that by varying the length of the linker molecule, and, accordingly, the distance between the two active molecules, the affinity of the tethered inhibitor compound for the NAD synthetase enzyme will also vary.
In practice of the invention relating to the design of novel NAD synthetase enzyme inhibitor compounds, a software program can be utilized which facilitates the prediction of the binding affinities of molecules to proteins so as to allow identification of commercially available small molecules with the ability to bind to at least one receptor subsite in the bacterial NAD synthetase enzyme. An example of one such computer program is DOCK, available from the Department of Pharmaceutical Chemistry at the University of California, San Francisco. DOCK evaluates the chemical and geometric complementarity between a small molecule and a macromolecular binding site. However, such a program would be useless in the design of a bacterial NAD synthetase enzyme inhibitor in the absence of complete information regarding the enzyme""s structure and the chemical makeup of the receptor sites, identified and disclosed fully for the first time herein.
With this invention, the crystal structure of one type of bacterial NAD synthetase enzyme e.g., B. subtilis has been for the first time identified fully. The x-ray crystal structure of NAD synthetase enzyme from B. subtilis had been reported in the literature. (M. Rizzi et al., The EMBO Journal, 15, 5125, (1996); M. Rizzi et al., Structure, 1129 (1998)). This was accomplished in free form and in complex with ATP and Mg+2 at 2.6 and 2.0 xc3x85, respectively. This structure contained the hydrolyzed form of ATP, namely AMP and Ppi, in the ATP binding site and ATP was present in the NaAD binding site. However, the prior art was not able to obtain the structure of the enzyme complex containing NaAD due to technical problems that precluded full identification. Without the structure of the enzyme complex containing NaAD, the structure-based drug design targeted to NAD synthetase enzyme of the present invention could not be developed.
In order to carry out structure-based drug design targeted to bacterial NAD synthetase enzyme, the structure of the enzyme in complex with all substrates, including NaAD has been solved herein. The additional structural information obtained in this invention for the first time clearly defined the interactions between NaAD and the enzyme, which provided information important for guiding combinatorial library design and inhibitor identification. Schematic drawings of crystal structures of the open and blocked receptor/catalytic sites of B. subtilis are set out previously in FIGS. 2 and 4.
The invention utilizes two approaches reported in the literature (for other biological targets) to help identify lead compounds. (1) Once the structure of a bacterial NAD synthetase catalytic site was identified, the software DOCK (I. D. Kunz et al., J. Mol. Biol., 161, 269-288 (1982)) was utilized to search the Available Chemicals Directory database and computationally score the relative binding affinities for each structure. Based on these results and structural information regarding substrate binding, commercially available compounds were selected for purchase and subsequent enzyme kinetics evaluation. Such database searching strategies in drug discovery are now commonly used by those of skill in the art of drug design. (D. T. Manallack, Drug Discovery Today, 1, 231-238 (1996)). (2) Using the results of biological screening for selected commercially available compounds to identify biologically active molecules, the inventors then designed a combinatorial library consisting of xe2x80x9ctethered dimersxe2x80x9d to rapidly identify more effective inhibitors of NAD synthetase enzyme as antibacterial agents. The use of xe2x80x9ctethered dimersxe2x80x9d to enhance the binding affinity of two moderately effective small molecule ligands that interact in the same binding site has been previously described in the literature. (S. B. Stuker, P. J. Hejduk, R. P. Meadows, and S. W. Fesik, Science, 274, 1531-1534 (1996)). However, this invention involves the first and, therefore, a novel application of database searching coupled with a combinatorial tethered dimer approach that was guided by the structure of and targeted to the bacterial NAD synthetase enzyme.
Examples from the top scoring small molecules as determined by, for example, DOCK, are preferably pre-screened using in vitro enzyme assays as further described herein. As a significant aspect of the invention herein, the preferred screening method utilized should allow the rapid screening of large numbers of compounds for inhibitory activity. In a preferred method of the present invention, the small molecule inhibitor candidate for each site that is most promising as an active molecule, as identified by DOCK (or other programs known to one of skill in the art) and the prescreening method herein, or that were designed based upon the substrate protein complex structure, were synthesized according to the methods disclosed herein below.
In one embodiment, the active molecules are chemically tethered to one another by means of a linker compound. In a further embodiment, the linker comprises one or more CH2 or other groups, using a variety of tether lengths, preferably 1 to 12 nonhydrogen atoms, more preferably 3 to 10 nonhydrogen atoms, further more preferably 5 to 9 nonhydrogen atoms and, still more preferably, 6 to 9 nonhydrogen atoms.
In another embodiment of the present invention, the novel compounds with preferred structures determined from the methods described above are synthesized by means of rapid, solution phase-parallel synthesis of the tethered dimers compounds in a combinatorial fashion. One of skill in the art will recognize such techniques. For each class of dimeric compounds designed in accordance with the invention herein, a novel synthetic strategy was developed to allow variation in the length of the linking group through which the active molecules are joined. These synthetic strategies are set forth herein as Schemes 3 through 7 and in Examples 1 through 5 below. Use of the preferred method of variable linkage greatly increases the number of different tethered dimeric compounds that can be produced from a single pair of the same or different active molecules. The active molecules specifically disclosed herein may be used, as well as any pharmaceutically acceptable salts thereof.
As noted, pharmaceutically acceptable salts of the compounds set out herein below are also contemplated for use in this invention. Such salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. The reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0xc2x0 C. to about 100xc2x0 C., preferably at room temperature. The molar ratio of compounds of structural formula (I) to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material-a particular preferred embodiment-the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt. When calcium salts are prepared, approximately one-half a molar equivalent of base is used to yield a neutral salt, while for aluminum salts, approximately one-third a molar equivalent of base will be used.
Similarly, salts of aliphatic and/or aromatic amines are also contemplated for use in this invention. A variety of pharmaceutically acceptable salts may be prepared by any of several methods well known to those skilled in the art. Such methods include treatment of a free aliphatic or aromatic amine with an appropriate carboxylic acid, mineral acid, or alkyl halide, or by conversion of the ammonium salt to another form using ion exchange resins.
Compounds prepared in accordance with the design and synthesis methods of this invention are especially attractive because they may preferably be further optimized by incorporation of substituents on either the active molecule and/or the linking group. These latter modifications can also preferably be accomplished using the combinatorial methods disclosed herein.
In a further embodiment of the present invention, selected novel compounds whose structures are designed by the above methods are synthesized individually using a novel strategy that allows variation in the length of the linking group. An example of a route preferably utilized to synthesize one class of dimers according to the present invention, using a single pair of active molecules, is summarized below in Scheme 3. 
In a preferred embodiment, the invention provides a method of making a bacterial NAD synthetase inhibitor compound comprising the steps of:
a. alkylating 5-nitroindole with 6-bromohexyl acetate to form a 6-[N-(5-nitroindolyl)]hexyl acetate;
b. hydrolyzing the 6-[N-(5-nitroindolyl)]hexyl acetate to form 6-[N(5 nitroindolyl)]hexan-1-ol;
c. esterifying the 6-[N-(5-nitroindolyl)]hexan-1-ol with nicotinic acid to form 6[N-(5-nitroindolyl)]hexyl nicotinate; and
d. N-methylating the 6-[N-(5-nitroindolyl)]hexyl nicotinate.
The following compounds were prepared according to Scheme 3 above, wherein n represents the number of linker groups tethering the two active molecules together.
Examples of additional preferred synthetic procedures utilized for preparing the library of the present invention are provided in Schemes 4-7. In Schemes 4-7, it is preferable to utilize combinational methods of synthesis using, for example, parallel solution phase synthetic techniques. One of skill in the art will readily recognize the manner in which the synthetic pathways disclosed below may be varied without departing from the novel and unobvious aspects of the invention. 
In a preferred embodiment, the invention provides a method of synthesizing a NAD synthetase inhibitor compound from the route set out in Scheme 4 above, comprising the steps of:
a. alkylating 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol;
b. reacting the indole alkyl alcohol with the appropriate reagent to form an indole alkyl ester; and
c. N-methylating the indole alkyl ester.
In yet another embodiment, the invention provides a method of making a NAD synthetase inhibitor compound from the route set out in Scheme 4 above comprising the steps of:
a. alkylating 5-nitroindole with bromoalkyl acetate wherein the indole alkyl acetate is converted to indole alkyl alcohol;
b. reacting the indole alkyl alcohol with the appropriate reagent to form an indole alkyl ester, and
c. reacting the indole alkyl alcohol with mesyl chloride followed by reaction with an amine to generate an ammonium product. 
In yet a further, still preferred, embodiment, the invention provides a method of making a NAD synthetase inhibitor from the route set out in Scheme 5 above, comprising the steps of:
a. reacting indole carboxylic acid with the appropriate reagent to provide an indole carboxylate methyl ester or an indole benzyl carboxylate ester;
b. V-alkylating the indole carboxylate methyl ester or the indole carboxylate benzyl ester with bromoalkyl acetate;
c. reacting the material from step b above with the appropriate reagent to form an indolealkyl alcohol;
d. coupling the indolealkyl alcohol with an aromatic amine; and
e. reacting the indolealkyl alcohol with the appropriate reagent to convert the methyl or benzyl indolecarboxylate to the respective indole carboxylic acids. 
In a further preferred embodiment, the invention provides a method of making a NAD synthetase inhibitor from the route set out in Scheme 6 above, comprising the steps of:
a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline;
b. reacting the 2-bromo-R1-substituted-aniline or the 2-bromo-R2-substituted-aniline using a Heck coupling reaction to form an alkyne-substituted aniline;
c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol;
d. quaternizing the indole alcohol with an amine;
e. reacting the indole alcohol with methansulfonyl chloride to provide an indole mesylate; and
f. reacting the indole mesylate with a carboxylic acid to form an indole ester.
In yet another preferred embodiment, the invention provides a method of making a NAD synthetase inhibitor compound from the route set out in Scheme 6 above, comprising the steps of:
a. brominating an aniline with N-bromosuccinimide to form a 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline;
b. reacting the 2-bromo-R1-substituted-aniline or a 2-bromo-R2-substituted-aniline using a Heck coupling reaction to form an alkyne-substituted aniline;
c. reacting the alkyne-substituted aniline using a cyclization reaction to form an indole alcohol;
d. quaternizing the indole alcohol with an amine;
e. reacting the indole alcohol with triflouromethylsulfonic anhydride to provide a triflate; and
f. reacting the indole triflate with an amine to form an indole alkylammonium product. 
In a preferred embodiment, the invention provides a method of synthesizing a NAD synthetase inhibitor compound from the route set out in Scheme 7 above, comprising the steps of:
a. alkylating a phenol with 7-bromo-1-heptanol to provide 7-(phenyloxy)-1-heptanol;
b. mesylating 7-(phenyloxy)-1-heptanol to provide 7-(phenyloxy)-1-heptyl methanesulfonate;
c. esterifying 7-(phenyloxy)-1-heptyl-methanesulfonate to provide 7-(phenyloxy)-1-heptyl nicotinate; and
d. n-methylating 7-(phenyloxy)-1-heptyl nicotinate to provide [7-(phenyloxy)-1-heptyl-(N-methyl)nictotinate]iodide.
In a preferred embodiment, the invention provides a compound having the general structure of Structure 2: 
wherein:
n is an integer of from 1 to 12, R1-R7 each, independently, is an H, an unsubstituted or a substituted cyclic or aliphatic group, a branched or an unbranched group, and wherein the linker is a cyclic or aliphatic, branched or an unbranched alkyl, alkenyl, or an alkynyl group and wherein the linker may also contain heteroatoms. By heteroatoms, it is meant that one or more atoms is an element other than carbon.
R1-R7, may also be one of the following groups: an H, alkyl, alkenyl, alknyl, or an aryl. R1-R7, may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. Note that n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, e.g., in this example denoted xe2x80x9caryl,xe2x80x9d moieties may be the same or different.
In a further embodiment, the invention provides a compound of Structure 4: 
wherein:
X is a C, N, O or S within a monocyclic or bicyclic moiety, A and B represent the respective sites of attachment for the linker, n is an integer of from 1 to 12, R1-R7 each, independently, is an H, an unsubstituted or a substituted cyclic group, or an aliphatic group, or a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic group or an aliphatic branched or unbranched alkyl, alkenyl or alkynyl group, and wherein the linker may also contain heteroatoms.
R1-R7 may also be one of the following groups: an H, alkyl, alkenyl, alkynyl, or an aryl group. R1-R7 may also be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. One of skill in the art would know what moieties are considered to constitute derivatives of these groups. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In a further embodiment, the invention provides a compound of Structure 6: 
wherein:
X is C, N, O or S, Y is C, N, O, S, carboxy, ester, amide, or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 1 to 12, and R1-R7 each, independently, is an H, unsubstituted or substituted cyclic group or an aliphatic group, a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl, or alkynyl group and wherein the linker may also contain heteroatoms.
R1-R7, may also be one of the following groups: an H, alkyl, alkenyl, alknyl, or an aryl. R1-R7, may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. Note that n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, e.g., in this example denoted xe2x80x9caryl,xe2x80x9d moieties may be the same or different.
In a further embodiment, the invention provides a compound of Structure 7: 
wherein:
X is C, N, O or S, Y is C, N, O, S, carboxy, ester, amide, or ketone, A and B represent the respective sites of attachment for a linker, n is an integer of from 1 to 12, and R1-R6 each, independently, is an H, unsubstituted or substituted cyclic group or an aliphatic group, a branched or an unbranched group, and the linker is a saturated or unsaturated cyclic or aliphatic group, branched or unbranched alkyl, alkenyl, or alkynyl group and wherein the linker may also contain heteroatoms.
R1-R6 may also be one of the following groups: an H, alkyl, alkenyl, alknyl, or an aryl. R1-R6, may further be a hydroxyl, ketone, nitro, amino, amidino, guanidino, carboxylate, amide, sulfonate, or halogen or the common derivatives of these groups. Note that n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9. The tethered active molecule, e.g., in this example denoted xe2x80x9caryl,xe2x80x9d moieties may be the same or different.
In a further embodiment, the invention provides a compound of Structure 8: 
wherein:
n is an integer of from 1 to 12, R1 is an H, methoxy, benzyloxy, or nitro and R2 is 3-pyridyl, N-methyl-3-pyridyl, 3-quinolinyl, N-methyl-3-quinolinyl, 3-(dimethylamino)phenyl, 3-(trimethylammonio)phenyl, 4-(dimethylamino)phenyl, 4-(trimethylammonio)phenyl, 4-(dimethylamino)phenylmethyl, or 4-(trimethylammonio)phenylmethyl.
In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In a further embodiment, the invention provides a compound of Structure 10: 
wherein:
n is an integer of from 1 to 12, R1 is an H, CO2H, xe2x80x94OCH3, or xe2x80x94OCH2Ph, R2 is H, CO2H, or CHxe2x95x90CHCO2H, R3 is H or CO2H, and Y is N-linked pyridine-3-carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In a further embodiment, the invention provides a compound of Structure 12: 
wherein:
n is an integer of from 1 to 12, R1 is H, F, or NO2, R2 is H, CH3, CF3, NO2, phenyl, n-butyl, isopropyl, F, phenyloxy, triphenylmethyl, methoxycarbonyl, methoxy, carboxy, acetyl, or benzoyl, R3 is H or CF3 and Y is N-linked pyridine-3-carboxylic acid, N-linked pyridine, N-linked quinoline, or N-linked isoquinoline. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In a further embodiment, the invention provides a compound of Structure 14: 
wherein:
n is an integer of from 1 to 12, R1 is H, phenyloxy, isopropyl, acetyl, or benzoyl, R2 is H or CF3, and Y is 3-(dimethylamino)phenyl, 3-(trimethylammonio)phenyl, 4-(dimethylamino)phenyl, 4-(trimethylammonio)phenyl, 2-(phenyl)phenyl, diphenylmethyl, 3-pyridyl, 4-pyridyl, or pyridine-3-methyl. In further embodiments, n may also be an integer of from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In a further embodiment, the invention provides a compound of Structure 16: 
wherein R is H or CO2CH3 and n is an integer of from 1 to 4, more preferably 2 to 3, and even more preferably, n is 3.
In a further embodiment, the invention provides a compound of Structure 18: 
wherein R is H or CO2CH3 and n is an integer of from 1 to 4, more preferably 2 to 3, and even more preferably, n is 3.
In further preferred embodiments of the invention herein, compounds of the structures denoted in Tables 102-128 as Compounds 1-274 were synthesized utilizing the methods disclosed herein. For Compounds 1-274, structures denoted in FIG. 6 as Fragments I-X each represent an active molecule, as defined previously herein, which can be included in the compounds of the present invention as further described in the respective Tables. In Fragments I-X of FIG. 6, the point of attachment for the linker compound is at the nitrogen.
In the chemical structures that follow, and as intended for the compounds of this invention, the symbol Xxe2x88x92 and Pxe2x88x92 designate generally the presence of an anion. As contemplated by the present invention; the type of anion in the compounds of this invention is not critical. The anions present in the compounds of this may be comprised of any such moieties known generally to one of skill in the art or that follow from the synthesis methods disclosed herein. 
FIG. 6: FRAGMENTS UTILIZED IN COMPOUNDS 1-274
In preferred embodiments of the invention herein, the compounds of the present invention correspond to Structure 100: 
wherein Rxe2x80x2 is: 
and n is an integer of from 1 to t2. N may also be from 3 to 10, more preferably 5 to 9 and, still more preferably 6 to 9.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 100 and as further defined in Table 100. For those compounds that correspond to Structure 100, n may also be an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6 and n indicates the number of linker groups separating the two tethered active molecule groups in the compound.
As set out below in relation to Compounds 25-274, Fragments A-G are set out in FIG. 8. The group denoted R in A-G of FIG. 8 can be a benzyl group, a methyl group or a hydrogen. The point of attachment of the linker group to. Fragments A-G is at the nitrogen group.
In one embodiment, the compounds of the present invention correspond to compounds of Structure 101. For those compounds that correspond to Structure 101, n is an integer of from 1 to 12, more preferably from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. The point of attachment of the linker group for both R1 and Rxe2x80x2 is at the respective nitrogen groups of each illustrated fragment. 
wherein Rxe2x80x2 is: 
wherein R1 is: 
wherein the R group in Fragments A-G is a benzyl group, a methyl group or a hydrogen.,
In one embodiment of the invention herein, the compounds of the present invention may include the Fragments illustrated below in FIG. 8. 
FIG. 8: FRAGMENTS A-G IN COMPOUNDS 25-274
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 102. For those compounds that correspond to Structure 102, n is an integer of from i to 12, from 3 to 10, more preferably from 5 to .9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 102, as further set out in Table 102. 
In the above table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, A corresponds to a fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and A in the respective compounds. Groups I, II, VII, VIII each have a benzyl group and Groups I*, III*, VII*, VIII* each have a hydrogen, respectively in the position designated R in Fragment A of FIG. 8.
In further preferred embodiments of the invention herein the compounds of the present invention correspond to the structures set out in Structure 104. For those compounds that correspond to Structure 104, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 104, as further set out in Table 104. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, B corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and B in the respective compounds. Groups I, VII, VIII each have a benzyl group and Groups I*, VII*, VIII* each have a hydrogen, respectively, in the-position designated R in Fragment B of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 106. For those compounds that correspond to Structure 106, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 106, as further set out in Table 106. 
In the above table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, C corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and C in the respective compounds. Groups I, II, VII, VIII each have a benzyl group and Groups I*, II*, VII*, VII* each have a hydrogen, respectively, in the position designated R in Fragment C of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 108. For those compounds that correspond to Structure 108, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 108, as further set out in Table 108. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, D corresponds to a fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and D in the compound. Groups I, VII, VIII each have a benzyl group and Groups I*, VII*, VIII* each have a hydrogen, respectively, in the position designated R in Fragment D of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 110. For those compounds that correspond to Structure 110, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 110, as further set out in Table 110. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, E corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and E in the respective compounds. Groups I, VII, VIII each have a benzyl group and Groups I*, VII*, VIII* each have a hydrogen, respectively in the position designated R in Fragment E of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 112. For those compounds that correspond to Structure 112, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 112, as further set out in Table 112. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, F corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and F in the respective compounds. Groups I, VI, VIII each have a benzyl group and Groups I*, VII*, VIE* each have a hydrogen, respectively, in the position designated R in Fragment F of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 114. For those compounds that correspond to Structure 114, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 114, as further set out in Table 114. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, G corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and G in the respective compounds. Groups I, VII, VIII each have a benzyl group and Groups I*, VII*, VIII* each have a hydrogen, respectively, in the position designated R in Fragment G of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 116. For those compounds that correspond to Structure 116, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 116, as further set out in Table 116. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, A corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and A in the respective compounds. Groups I, II each have a methyl group and Groups I*, II* each have a hydrogen, respectively, in the position designated R in Fragment A of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 118. For those compounds that correspond to Structure 118, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 118, as further set out in Table 118. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, B corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and B in the respective compounds. Groups I, II each have a methyl group and Groups I*, III* each have a hydrogen, respectively, in the position designated R in Fragment B of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 120. For those compounds that correspond to Structure 120, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 120, as further set out in Table 120. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, C corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and C in the respective compounds. Groups I, II each have a methyl group and Groups I*, II* each have a hydrogen, respectively, in the position designated R in Fragment C of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 122. For those compounds that correspond to Structure 122, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9.1In further embodiments, the compounds herein correspond to Structure 122, as further set out in Table 122. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, D corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups. Rxe2x80x2 and D in the respective compounds. Groups I, II each have a methyl group and Groups I, III each have a hydrogen, respectively, in the position designated R in Fragment D of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 124. For those compounds that correspond to Structure 124, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 124, as further set out in Table 124. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, E corresponds to a Fragment as previously defined in FIG. 8, and h indicates the number of linker groups separating Groups Rxe2x80x2 and E in the respective compounds. Groups I, II each have a methyl group and Groups I*, III* each have a hydrogen, respectively, in the position designated R in Fragment E of FIG. 8.
In further preferred embodiments of the invention herein, the compounds of the present invention correspond to the structures set out in Structure 126. For those compounds that correspond to Structure 126, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 126, as further set out in Table 126. 
In the above Table Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, F corresponds to a Fragment as previously defined in FIG. 8, and n indicates the number of linker groups separating Groups Rxe2x80x2 and F in the respective compounds. Groups I, II each have a methyl group and Groups I*, III* each have a hydrogen, respectively, in the position designated in Fragment F of FIG. 8.
In further embodiments of the invention herein, the compounds of the present invention corresponds to the structures set out in Structure 128. For those compounds that correspond to Structure 128, n is an integer of from 1 to 12, from 3 to 10, more preferably from 5 to 9, and still more preferably from 6 to 9. In further embodiments, the compounds herein correspond to Structure 128, as further set out in Table 128. 
In the above Table, Rxe2x80x2 corresponds to a Fragment as previously defined in FIG. 6, G corresponds to, a Fragment as previously defined in FIG. 6, and n indicates the number of linker groups separating Groups Rxe2x80x2 and G in the respective compounds. Groups I, II each have a methyl group and Groups I*, III* each have a hydrogen, respectively, in the position designated R in Fragment G of FIG. 8.
As used herein, the following terms are defined as follows: Ph: phenyl; 1-propylxe2x95x90isopropyl; OPhxe2x95x90O-Phenyl; and diNO2xe2x95x90dinitric.
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 130 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. Further preferred embodiments of the compounds corresponding to Structure 130 are set out in Table 130. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 132 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 5-H, 6-CF3, 5-CH3, 5,7-diF, 5,7-diNO2, 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO2, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF3, 5-COCH3, 5-OCH3, 5-COOCH, or 5-COOH.
Further preferred embodiments of the compounds corresponding to Structure 132 are set out in Table 132. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 134 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-H, 6-CF3, 5-CH3, 5,7-diF, 5,7-diNO2, 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO2, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF3, 5-COCH3, 5-OCH3, 5-COOCH3, or 5-COOH. Further preferred embodiments of the compounds corresponding to Structure 134 are set out in Table 134. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 136 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-H, 6-CF3, 5-CH3, 5,7-diF, 5,7-diNO2, 5-Butyl, 5-iPropyl, 5-Phenyl, 5-NO2, 5-Trityl, 5-F, 5-OPh, 5-COPh, 5-CF3, 5-COCH3, 5-OCH3, 5-COOCH3, or 5-COOH. Further preferred embodiments of the compounds corresponding to Structure 136 are set out in Table 136. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 138 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-CF3, 5-OPh, 5-iPropyl, 5-COCH3, or 5-COPh and Y is 3-N,N-dimethylaminophenyl (3-N,N-diCH3), 4N,N-dimethylaminophenyl (4-N,N-diCH3), or 2-Ph. Further preferred embodiments of the compounds corresponding to Structure 138 are set out in Table 138. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 140 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 5-CF3, 5-OPh, 5-iPropyl, 5-COCH3 or 5-COPh, and Z is CH(Ph)2 or 3-Pyridyl. Further preferred embodiments of the compounds corresponding to Structure 140 are set out in Table 140. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 142 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-iPropyl, 5-COCH3, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 142 are set out in Table 142. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 144 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-iPropyl, 5-COCH3, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 144 are set out in Table 144. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 146 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9. Further preferred embodiments of the compounds corresponding to Structure 146 are set out in Table 146. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 148, as further defined in Table 148. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 150 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.
Further preferred embodiments of the compounds corresponding to Structure 150 are set out in Table 150. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 152 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.
Further preferred embodiments of the compounds corresponding to Structure 152 are set out in Table 152. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 154 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein Z is CH(DiPh), 4-(N,N-dimethylamino)phenyl, CH2CH2-(3-pyridyl), or (2-phenyl)-phenyl. Further preferred embodiments of the compounds corresponding to Structure 154 are set out in Table 154. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 156 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 156 are set out in Table 156. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 158 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 158 are set out in Table 158. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 160 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 160 are set out in Table 160. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 162 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 162 are set out in Table 162. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 164 wherein n is an Integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 164 are set out in Table 164. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 166 wherein n is an integer of from i to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH, or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 166 are set out in Table 166. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 168 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 168 are set out in Table 168. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 170 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 or xe2x80x94OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 170 are set out in Table 170. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 172 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 and xe2x80x94OCH2 Ph. Further preferred embodiments of the compounds corresponding to Structure 172 are set out in Table 172. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 174 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is xe2x80x94OCH3 and xe2x80x94OCH2 Ph. Further preferred embodiments of the compounds corresponding to Structure 174 are set out in Table 174. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 176 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein Z is 3-quinoline, 3-(N,N-dimethylamino)phenyl, or 44N,N-dimethylamino)phenyl. Further preferred embodiments of the compounds corresponding to Structure 176 are set out in Table 176. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 178 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6to 9.
Further preferred embodiments of the compounds corresponding to Structure 178 are set out in Table 178. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 180 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.
Further preferred embodiments of the compounds corresponding to Structure 180 are set out in Table 180. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 182 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9.
Further preferred embodiments of the compounds corresponding to Structure 182 are set out in Table 182. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 184 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF3, 5-OPh, 5-CH(CH3)2, 5-COCH, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 184 are set out in Table 184. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 186 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-CH(CH3)2, 5-COCH, or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 186 are set out in Table 186. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 188 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF3, 5-OPh, 5-CH(CH3)2, 5-COCH3 or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 188 are set out in Table 188. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 190 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is 6-CF3, 5-OPh, 5-CH(CH3)2, 5-COCH3 or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 190 are set out in Table 190. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 192 wherein n is an integer of from 1l to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein and R is 6-CF3, 5-OPh, 5-CH(CH3)2, 5-COCH3 or 5-COPh. Further preferred embodiments of the compounds corresponding to Structure 192 are set out in Table 192. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 194 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and R1 is an H or xe2x80x94OCH2Ph and R2 is H or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 194 are set out in Table 194. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 196 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R1 is an H or a xe2x80x94OCH2Ph and R2 is H or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 196 are set out in Table 196. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 198 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R1 is an H, xe2x80x94OCHPh or xe2x80x94OCPh3 and R2 is H, or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 198 are set out in Table 198. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 200 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R1 is H or a xe2x80x94OCH7Ph and R2 is H or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 200 are set out in Table 200. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 202A. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 202A wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably from 6 to 9 and wherein R is H; 4-NO2; 2-CONHPh; 2-NO2; 4-[1xe2x80x2(4xe2x80x2-acetylpiperazine)]; 2-COCH3; 3-OCOCH3; 3-OCH3; 4-COCH3; 3-OCOPh; 2-CONH2; 4-CHxe2x95x90CHCOCH3; 4-OCOPh; 4-CHxe2x95x90CHCOPh; 4-{CO-3xe2x80x2[2xe2x80x2-butylbenzo(b)furan]}; 3-NO2; 4-[5xe2x80x2(5xe2x80x2-phenylhydantoin)]; 2-CHxe2x95x90CHCOPh; 2-OCH3; 4-COPh; 4-CONH2; 3-COCH3; 4-OPh; 4-(N-Phthalimide); 3-(N-Morpholine); 2-(N-pyrrolidie); 2-(N-Morpholine); or 4-OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 202 are set out in Table 202.
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 204A wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is 4-NO2; 2-CONHPh; 2-NO2; 4-[1xe2x80x2(4xe2x80x2-acetylpiperazine)]; 2-COCH3; 3-OCOCH3; 3-OCH3; 4-COCH3; 3-OCOPh; 2-CONH2; 4-CHxe2x95x90CHCOCH3; 4-OCOPh; 4-CHxe2x95x90CHCOPh; 4-{CO-3xe2x80x2[2xe2x80x2-butylbenzo(b)furan]}; 3-NO2; 4-[5xe2x80x2-(5xe2x80x2-phenylhydantoin)]; 4-CHxe2x95x90CHCOPh; 2-OCH3; 4-COPh; 4-CONH2; 3-COCH3; 4OPh; 4-(N-phthalimide); 3-(N-morpholine); 2-(N-pyrrolidine); 2-(N-morpholine); or 4-OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 204 are set out in Table 204. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 206 wherein n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is H; 4-NO2; 2-CONHPh; 2-NO2; 2-COCH3; 3-OCH3; 4-COCH3; 3-OCOPh; 2-CONH2; 4-CHxe2x95x90CHCOCH3; 4-OCOPh; 4-CHxe2x95x90CHCOPh; 4-{CO-3xe2x80x2[2xe2x80x2-butylbenzo(b)furan]}; 3-NO2; 2-CHxe2x95x90CHCOPh; 2-OCH3; 4-COPh; 3-COCH3; 4-OPh; 4-(N-phthalimide); or 4-OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 206 are set out in Table 206. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 208 wherein, n is an integer of from 1 to 12, more preferably, from 3 to 10, more preferably from 5 to 9 and, still more preferably, from 6 to 9 and wherein R is 4-NO2; 2-CONHPh; 2-NO2; 2-COCH3; 3-OCH3; 4-OCOPh; 2-CONH2; 4-CHxe2x95x90CHCOCH3; 4-OCOPh; 4-CHxe2x95x90CHCOPh; 4-{CO-3xe2x80x2[2xe2x80x2-butylbenzo(b)furan]}; 3-NO2; 2-CHxe2x95x90CHCOPh; 2-OCH3; 4-COPh; 3-COCH3; 4-OPh; 4-(N-phthalimide); 3(N-morpholine); 2-(N-morpholine); or 4-OCH2Ph. Further preferred embodiments of the compounds corresponding to Structure 208 are set out in Table 208. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 210 wherein R is NH2; NMe2; NMe3.I; NH2.HCl; NME2.HCl. Further preferred embodiments of the compounds corresponding to Structure 210 are set out in Table 210. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 212 wherein R1 is PhCONH or Ph3C and Rxe2x80x3 is H or COOCH3. Further preferred embodiments of the compounds corresponding to Structure 212 are set out in Table 212. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of the Structure 214 wherein R is 4-hydroxyphenyl or 3-hydroxy-4-methylphenyl. Further preferred embodiments of the compounds corresponding to Structure 214 are set out in Table 214. 
In further embodiments, the compounds of the present invention preferably correspond to compounds of Structure 216 wherein Rxe2x80x2 is PhCONH and and Rxe2x80x3 is H or COOCH3 and n=7 or 8. Further preferred embodiments of the compounds corresponding to Structure 216 are set out in Table 216. 
In a particularly preferred embodiment of the invention herein, the present invention comprises compounds of the structures in Table 301 below.
In a further preferred embodiment, the present invention comprises one or more compounds from Table 302, below.
In a further preferred embodiment, the present invention comprises one or more compounds from Table 303, below.
The compounds of the invention may be readily synthesized using techniques generally known to synthetic organic chemists. Suitable experimental methods for making and derivatizing aromatic compounds are described, for example, methods for making specific and preferred compounds of the present invention are described in detail in Examples 1 to 4 below.
This invention preferably further provides a method of generating a library comprising at least one bacterial NAD synthetase enzyme inhibitor compound comprising the steps of:
a. obtaining the crystal structure of a bacterial NAD synthetase enzyme;
b. identifying one or more sites of catalytic activity on the NAD synthetase enzyme;
c. identifying the chemical structure of the catalytic sites on the NAD synthetase enzyme;
d. selecting one or more active molecule compounds that will demonstrate affinity for at least one of the catalytic sites on the NAD synthetase enzyme;
e. synthesizing one or more dimeric compounds comprised of at least one active molecule wherein the active molecule compound are joined by means of n linker compounds and wherein n is an integer of from 1 to 12, and
f. screening the one or more compounds for NAD synthetase inhibitor activity.
The library further comprises one or more compounds set forth in Table 301 above. In one embodiment, a library of compounds according to the invention herein preferably includes compounds of the structures set out in structures 1 to 1106 above. Further preferably, the library comprises a compound of Structure 2, still preferably, Structure 4, further preferably, Structure 6, and further preferably, Structure 7. In further preferred embodiments, the library comprises at least one compound of Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.
In another preferred embodiment of the invention herein, the one or more dimeric compounds comprise at least two active molecules. Still preferably, the active molecules are the same. Alternatively, it is preferable that the active molecules are different.
In the invention herein, a software program that predicts the binding affinities of molecules to proteins is utilized in the active molecule selection step. Further preferably, a software program that evaluates the chemical and geometric complementarity between a small molecule and macromolecular binding site is utilized in the active molecule selection step.
In yet another preferred embodiment, the compounds are synthesized utilizing a rapid, solution phase parallel synthesis and wherein the compounds are generated in a combinatorial fashion.
In a preferred embodiment, the invention provides a method of treating or preventing a microbial infection in a mammal comprising administering to the mammal a treatment effective or treatment preventive amount of a bacterial NAD synthetase enzyme inhibitor compound. In a particularly preferred embodiment, the compound administered in the method is a compound as set out previously in Table 301. In another embodiment, invention herein preferably includes compounds 1 to 1106 above. Further preferably, the compound administered comprises at least one compound of Structure 2, still preferably, Structure 4, further preferably, Structure 6. In further preferred embodiments, the compounds administered in the method comprise compounds of Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.
In a preferred embodiment, the invention provides administering a broad spectrum antibiotic to a mammal in need of such treatment or prevention. In a further preferred embodiment, the microbial infection is a bacterial infection. In yet another embodiment of the invention, the bacterial infection is caused by a bacterium that is a gram negative or gram positive bacteria The bacterial infection may preferably be caused by an antibiotic resistant strain of bacteria.
Further provided by the invention herein is preferably a method of killing a prokaryote with an amount of prokaryotic NAD synthetase enzyme inhibitor compound to reduce or eliminate the production of NAD whereby the prokaryote is killed. A method of decreasing prokaryotic growth, comprising contacting the prokaryote with an amount of a prokaryotic NAD synthetase enzyme inhibitor effective to reduce or eliminate the production of NAD whereby prokaryotic growth is decreased is also provided. In the method of killing a prokaryote, as well as in the method of decreasing prokaryotic growth, the compound comprises one or more compounds of Table 301, Table 302 or Table 303. Still preferably, the invention comprises one or more of compounds 1 to 1106 above. Further preferably, the compound administered is a compound of Structure 2, still preferably, a compound of Structure 4, further preferably, Structure 6. In further preferred embodiments, the compounds administered in the methods compounds of Structure 7, Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.
In the method of killing a,prokaryote, as well as in the method of decreasing prokaryotic growth, the prokaryote is a bacterium. Further preferably, the bacterium is a gram negative or a gram positive bacteria. Still preferably, the prokaryote is an antibiotic resistant strain of bacteria.
Also in the method of killing a prokaryote, as well as in the method of decreasing prokaryotic growth, the NAD synthetase enzyme inhibitor is a compound that selectively binds with catalytic sites or subsites on a bacterial NAD synthetase enzyme to reduce or eliminate the production of NAD by the bacteria.
In the methods discussed above, the compound is preferably administered by oral, rectal, intramuscular, intravenous, intravesicular or topical means of administration. The compounds of this invention can be administered to a cell of a subject either in vivo or ex vivo. For administration to a cell of the subject in vivo, as well as for administration to the subject, the compounds of this invention can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, subcutaneous injection, transdermally, extracorporeally, topically, mucosally or the like.
Depending on the intended mode of administration, the compounds of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected composition, possibly in combination with a pharmaceutically acceptable carrier and, In addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
Parenteral administration of the compounds of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. As used herein, xe2x80x9cparenteral administrationxe2x80x9d includes intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes. One approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein. These compounds can be present in a pharmaceutically acceptable carrier, which can also include a suitable adjuvant. By xe2x80x9cpharmaceutically acceptable,xe2x80x9d it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
Routes of administration for the compounds herein are preferably in a suitable and pharmacologically acceptable formulation. When administered to a human or an animal subject, the bacterial NAD synthetase enzyme inhibitor compounds of the libraries herein are preferably presented to animals or humans orally, rectally, intramuscularly, intravenously, intravesicularly or topically (including inhalation). The dosage preferably comprises between about 0.1 to about 15g per day and wherein the dosage is administered from about 1 to about 4 times per day. The preferred dosage may also comprise between 0.001 and 1 g per day, still preferably about 0.01, 0.05, 0.1, and 0.25, 0.5, 0.75 and 1.0 g per day. Further preferably, the dosage may be administered in an amount of about 1, 2.5, 5.0, 7.5,10.0, 12.5 and 15.0 g per day. The dosage may be administered at a still preferable rate of about 1, 2, 3, 4 or more times per day. Further, in some circumstances, it may be preferable to administer the compound of the invention continuously, as with, for example, intravenous administration. The exact amount of the compound required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular compound used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every compound. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
If ex vivo methods are employed, cells or tissues can be removed and maintained outside the subject""s body according to standard protocols well known in the art. The compounds of this invention can be introduced into the cells via known mechanisms for uptake of small molecules into cells (e.g., phagocytosis, pulsing onto class I MHC-expressing cells, liposomes, etc.). The cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
It is further provided a method of disinfecting a material contaminated by a microbe, comprising contacting a contaminated material with a bacterial NAD synthetase enzyme inhibitor compound in an amount sufficient to kill or deactivate the microbe. In yet another embodiment, the compound utilized for contacting comprises one or more compounds of Table 301, Table 302 or Table 303. The compounds utilized for contacting may also comprise one or more of compounds 1 to 1106. Further preferably, the compound utilized for contacting is a compound of Structure2, still preferably, a compound of Structure 4, further preferably, Structure 6. In further preferred embodiments, the compounds utilized for contacting in the method comprise compounds of Structure 7, Structure 8, Structure 10, Structure 12, Structure 16 or Structure 18.
In yet a further embodiment of the invention herein, the compounds of the present invention are effective as disinfectant materials for, for example, hard or soft surfaces, fabrics, and other contaminated materials such as those in hospitals, households, schools, nurseries, and any other location. In yet another embodiment, the invention provides a method for disinfecting comprising contacting a bacterial contaminated material with a bacterial NAD synthetase enzyme inhibitor compound.
In a further aspect of the invention, an in vitro xe2x80x9cone-at-a-timexe2x80x9d method of screening compounds for bacterial NAD synthetase enzyme inhibitory activity is provided. In a preferred embodiment, this in vitro method of screening compounds for such activity comprises the steps of preparing a solution comprising pure bacterial NAD synthetase enzyme, contacting the solution with the compounds set out herein, and determining the rate of the enzyme-catalyzed reaction. Preferably, measurement of the rate of enzyme-catalyzed reaction comprises a measure of NAD synthetase inhibitory activity. In a further embodiment the rate of enzyme-catalyzed reaction comprises a measure of antibacterial activity. In a still further embodiment, the rate of enzyme-catalyzed reaction corresponds to a measure of antimicrobial activity.
Preferably, the method of preparing the bacterial enzyme solution for use in the in vitro screening method comprises utilizing molecular biological methods to over-express bacterial NAD synthetase enzyme, for example from B. subtilis, in E. coli. One of skill in the art will recognize techniques useful for such a process. A particularly preferable method comprises: a) cloning the Out B gene encoding NAD synthetase enzyme and over-expressing the gene in E. coli; b) purifying the cloned and over-expressed gene by ion-exchange; c) purifying further the enzyme material from step b using ion-exchange methods; d) further purifying the material from step c using size exclusion chromatography wherein the bacterial NAD synthetase enzyme is essentially pure; and e) preparing an assay solution in quantities of about 10 to 15 mg pure bacterial NAD synthetase enzyme per liter of fermentation-broth. As used herein, xe2x80x9cessentially purexe2x80x9d means greater than about 90% purity, more preferably, greater than about 95% purity and, still more preferably, greater than about 99% purity.
In one embodiment of the in vitro screening method, the following procedure is utilized to measure the rate of enzyme catalyzed reaction. A solution of HEPPS, pH 8.5, with KCl is prepared containing the following species: ATP, NaAD, MgCl2, NH4Cl, ADH, and ETOH. A stock solution of test inhibitors is then prepared by dissolving solid samples into 100% DMSO. The test compound stock solution is then added to the mixture to give the final test compound concentrations. NAD synthetase enzyme solution is added, the mixture is mixed three times, and the absorbance at 340 nm is then monitored kinetically using an UV-Vis spectrophotometer. The initial kinetics trace after enzyme addition is then fit to a straight line using linear regression, with this rate is then compared to that of a control containing no inhibitor, using the following formula to calculate % Inhibition: {(Voxe2x88x92V)/Vo}*100%, where Vo is the rate of the reaction with no test compound present and V is the rate of the reaction with test the test compound added. Each compound is tested in triplicate, and the resulting values for % inhibition were averaged to give the listed value. IC50 (concentration needed to inhibit 50% of the test bacteria) values were obtained for select compounds by assaying six different concentrations of test compound, in triplicate, at concentrations between 0.0 and 2.0 mM, and plotting the resulting % inhibition values against the xe2x88x92LOG of the test compound dose to reveal the concentration at which 50% inhibition is observed.
Preferably, the in vitro method can also be adapted to allow screening for compounds with bacterial NAD synthetase enzyme inhibitory activity in other forms of bacteria, as well as other types of microbes. For example, the above-described procedure can be adapted to screen for inhibitory activity in at least the following bacteria types:
In a further embodiment of the in vitro screening method, the method can be used to screen existing compounds e.g., commercially available compounds, such as 5-nitroindole and N-methyl nicotinic acid. One of skill in the art will recognize the manner in which the designing and screening methods herein can be utilized to identify commercially available compounds, such as the previous non-exhaustive list, that will exhibit NAD synthetase enzyme inhibitory activity, both in bacteria and other microbes.
In order to test a library of NAD synthetase enzyme inhibitor compounds, such as those of the present invention, it is particularly preferable to utilize a method of rapid (high throughput) screening. To this end, the potential inhibitory activity of the library of synthetic compounds in one embodiment is assessed via a coupled enzymatic assay. The coupled assay involves two steps as summarized below. 
In order to rapidly measure the inhibitory activities of the compounds in the library, the invention provides a high through-put screening system (HTS system). The HTS system preferably utilizes an integrated robotic system that coordinates the functions of a liquid handler and a spectrophotometer. The robotic station is preferably responsible for the movement of all hardware and the integration of multiple stations on the work surface. The liquid handler is preferably programmed to perform all phases of liquid dispensing and mixing. The spectrophotometer is preferably equipped to monitor absorbance in a 96-well plate format.
In one embodiment, the assay is designed for a 96-well plate format reaction buffer containing HEPPS buffer, pH 8.5, MgCl2, NH4Cl2, KCl, NAAD, n-Octyl-D-Glucopyranoside, ethanol, NAD synthetase, and yeast alcohol dehydrogenase. At the next stage, the liquid handler dispenses DMSO (with or without inhibitor) into the reaction well. The liquid handler mixes these components utilizing a predefined mixing program. The reaction is initiated by the addition of a solution of ATP dissolved in buffer. The reaction is monitored by measuring the increase in absorbance at 340 nm. The linear portion of the reaction is monitored for a period of time. The initial velocity is determined using the software supplied with the spectrophotometer.
The compounds of the library herein are supplied as a stock with a concentration dissolved in 100% DMSO. An initial screen is conducted on all compounds using a 2 or 3 concentration screen. The 2 panel screen used concentrations of 0.2 mM and 0.1 mM for the compounds. The 3 panel screen used concentrations of 0.2 mM, 0.1 mM, and 0.05 mM. From the initial screen, xe2x80x9clead compoundsxe2x80x9d e.g., those compounds which demonstrated the greatest inhibitory capacity, are then preferably subjected to a wider screen of -concentrations (0.1 mM to 0.001 mM) to determine the apparent IC-50 values for each compound.
In still a further preferred embodiment of the invention herein, the high through-put method is utilized to screen commercially available compounds for bacterial NAD synthetase enzyme inhibitory activity. In an additional embodiment, the NAD synthetase enzyme inhibitor compounds are tested as inhibitors of bacterial growth against a variety of bacteria types.
In a further embodiment of the invention, compounds within the libraries of NAD synthetase inhibitor compounds are evaluated for antibacterial and antimicrobial activity. In one embodiment, compounds are preferably evaluated for their potential to inhibit the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus epidermitis. The inhibitors are preferably initially screened in duplicate at one concentration The test inhibitor compounds are prepared by dissolving the solid samples in DMSO. Aliquots from the inhibitor stocks are placed in sterile 96-well plates by the liquid handler discussed previously. Cultures of B. subtilis, P. aeruginosa and S. epidermitis are prepared in liquid broth (LB) media and incubated in an orbital shaker overnight. Dilutions (with LB media) of the overnight cultures are added to the 96-well plates containing the inhibitors. The plates are incubated and the absorbance measured at 595 nm in a plate reader.
In this embodiment of the invention, a diluted overnight culture without inhibitors serves as one of three controls in the experiments. A positive control, which includes an identical concentration of the drug Tobramycin as the inhibitors being tested, and a DMSO control are also performed during each inhibitor screen. The DMSO control was included for comparison with the control that contained no inhibitors.
Percent inhibition of each inhibitor was calculated by the following formula: {(ADxe2x88x92AI)/AD}*100; where AD=the absorbance at 595 nm of the DMSO control and AI=the absorbance of the inhibitor at 595 nm.
In a further embodiment, dose responses are performed on the compounds that inhibited greater than 85% in the initial screen. The dose responses consisted of 5 different concentrations (from 100 mMxe2x88x920.1 mM) of each inhibitor and the positive control Tobramycin. The cultures are prepared and grown in the same manner as the inhibitor screens and the same controls were included. The absorbance is measured every hour and a half during the six hours of growth. Percent inhibitions are calculated again for each concentration tested. The lowest concentration that resulted in an 85% inhibition or higher is termed the Minimum Inhibitory Concentration that inhibited bacterial growth 85% (MICe5).
When a NAD synthetase enzyme inhibitor compound of a library herein are to be administered to a humans or an animal e.g., a mammal, it is preferable that the compounds show little or no toxicity to the patient. Therefore, in one embodiment of the invention herein, the toxicities of the NAD-synthetase enzyme inhibitors are evaluated using human epithelial cells as set out in Example 11 below.